Assembly dynamics and the roles of FliI ATPase of the bacterial flagellar export apparatus (original) (raw)
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Journal of Bacteriology, 2012
The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH EN ) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH EN with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.
The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H + –protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na + increased it dramatically. Phenamil, a blocker of Na + translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na + concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na + channel. In wild-type cells, however, neither Na + nor phenamil affected protein export, indicating that the Na + channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na + concentration. For construction of the bacterial flagellum beyond the inner and outer membranes, the fla-gellar type III export apparatus transports fourteen flagellar proteins with their copy numbers ranging from a few to tens of thousands to the distal growing end of the flagellar structure. The export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. Here, we show that the export engine of the flagellar type III export apparatus is robust in maintaining its export activity against internal and external perturbations arising from genetic variations and/or environmental changes. When the cytoplasmic ATPase complex is absent, the export gate complex is able to utilize
FliH and FliI ensure efficient energy coupling of flagellar type III protein export in Salmonella
MicrobiologyOpen, 2016
For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross-complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH-fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH-fliI flhB(P28T) ΔflhA mutant. Pull-down assays revealed that the binding affinities of ...
Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export
Molecular Microbiology, 2010
FlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N-terminal transmembrane domain (FlhATM) and a C-terminal cytoplasmic domain (FlhAC). FlhAC is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhAC at 2.8 Å resolution. FlhAC consists of four subdomains (ACD1, ACD2, ACD3 and ACD4) and a linker connecting FlhAC to FlhATM. The sites of temperature-sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation sug-gested that FlhA C changes its conformation for its function. Molecular dynamics simulation demonstrated an open-close motion with a 5-10 ns oscillation in the distance between ACD2 and ACD4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhAC is essential for protein export.
FliI, a soluble ATPase component of the flagellar export apparatus; FliH and FliJ, soluble regulatory components of the export apparatus; FliC, flagellin, the major filament protein; FliS, chaperone for FliC; T3SS, type III secretion system; FlhA, FlhB, FliO, FliP, FliQ and FliR, membrane components of the flagellar export apparatus; FlgK and FlgL, hook-filament junction proteins; FlgN, chaperone for FlgK and FlgL; FliD, filament-cap protein; FliT, chaperone for FliD; FliN, a C-ring protein; Footnote: Complexes with defined stoichiometry (e. g. FliC:FliS) are indicated with a colon (:), while complexes with varying or unknown stoichiometry (e.g. FliI-FliJ-FliH) are indicated with hyphen (-).
Stoichiometry and turnover of the bacterial flagellar switch protein FliN
mBio, 2014
Some proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio o...
Biochemistry, 2012
The binding of the soluble cytoplasmic protein FliG to the transmembrane protein FliF is one of the first interactions in the assembly of the bacterial flagellum. Once established, this interaction is integral in keeping the flagellar cytoplasmic ring, responsible for both transmission of torque and control of the rotational direction of the flagellum, anchored to the central transmembrane ring on which the flagellum is assembled. Here we isolate and characterize the interaction between the N-terminal domain of Thermotoga maritima FliG (FliG N ) and peptides corresponding to the conserved C-terminal portion of T. maritima FliF. Using nuclear magnetic resonance (NMR) and other techniques, we show that the last ∼40 amino acids of FliF (FliF C ) interact strongly (upper bound K d in the low nanomolar range) with FliG N . The formation of this complex causes extensive conformational changes in FliG N . We find that T. maritima FliG N is homodimeric in the absence of the FliF C peptide but forms a heterodimeric complex with the peptide, and we show that this same change in oligomeric state occurs in full-length T. maritima FliG, as well. We relate previously observed phenotypic effects of FliF C mutations to our direct observation of binding. Lastly, on the basis of NMR data, we propose that the primary interaction site for FliF C is located on a conserved hydrophobic patch centered along helix 1 of FliG N . These results provide new detailed information about the bacterial flagellar motor and support efforts to understand the cytoplasmic ring's precise molecular structure and mechanism of rotational switching.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature
Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP-FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.